Use of fk506 for the treatment of pulmonary arterial hypertension

ABSTRACT

A method for reducing pulmonary hypertension in a mammal that employs FK506 is provided. In certain embodiments, the method comprises administering FK506 to a mammal having pulmonary arterial hypertension associated with defective MBPR2 signaling at a dosage sufficient to reduce blood pressure in the pulmonary artery of the mammal.

GOVERNMENT RIGHTS

This invention was made with Government support under contract HL089989awarded by the National Institutes of Health. The Government has certainrights in this invention.

BACKGROUND

Pulmonary artererial hypertension is abnormally high blood pressure inthe arteries of the lungs. Because symptoms may develop very gradually,patients may delay seeing a physician for years. Common symptoms areshortness of breath, fatigue, non-productive cough, angina pectoris,fainting or syncope, peripheral edema (swelling around the ankles andfeet), and rarely hemoptysis(coughing up blood).

SUMMARY

A method of reducing pulmonary arterial hypertension in a mammal thatemploys FK506 is provided. In certain embodiments, the method comprisesadministering FK506 to a mammal having pulmonary arterial hypertensionassociated with defective BMPR2 signaling at a dosage sufficient toreduce blood pressure in the pulmonary artery of the mammal. In somecases, the mammal is a mouse, rat or a human. In particular cases, themammal has hereditary pulmonary arterial hypertension caused by, forexample, a mutation in BMPR2, ALK1 or endoglin. In particular cases theFK506 may be administered at a dose that provides an FK506 serumconcentration of 0.05 to 1 ng/ml, e.g. 0.1-0.2 ng/ml.

DETAILED DESCRIPTION OF EXEMPLARY EMBODIMENTS

The method described herein is for treating pulmonary arterialhypertension in patients that have defect in BMPR2 (bone morphogeneticprotein receptor 2) signaling. Pulmonary arterial hypertension (PAH) isa progressive lung disorder which, untreated, often leads to death onaverage within a few years after being diagnosed. An increasingconstriction of the pulmonary circulation leads to increased stress onthe right heart, which can develop into right heart failure. Bydefinition, the mean pulmonary arterial pressure (mPAP) in a case ofchronic pulmonary hypertension is >25 mmHg at rest or >30 mmHg duringexertion, where the normal values are <20 mmHg. The pathophysiology ofpulmonary arterial hypertension is characterized by vasoconstriction andremodeling of the pulmonary vessels. In chronic PAH there isneomuscularization of initially unmuscularized pulmonary vessels, andthe vascular muscles of the already muscularized vessels increase incircumference. This increasing obliteration of the pulmonary circulationresults in progressive stress on the right heart, which leads to areduced output from the right heart and eventually ends in right heartfailure (Humbert et al., J. Am. Coll. Cardiol. 2004, 43, 13S-24S).

Defects in BMPR2 signaling may, for example, be caused by a mutation inBMPR2 (see accession no. 600799 in NCBI's OMIM database), a mutation inALK1 (the activin A receptor; see accession no. 601284 in NCBI's OMIMdatabase) or a mutation in endoglin (see accession no. 131195 in NCBI'sOMIM database). A defect in BMPR2 signaling can be detected, forexample, by measuring the expression of Id1 (inhibitor ofdifferentiation 1) mRNA or protein, which is a well-known downstreamread-out for BMPR2 signaling. Decreased BMPR2 signaling decreases theamount of Id1 in pulmonary artery smooth muscle cells. In particularcases, the pulmonary arterial hypertension may be hereditary pulmonaryarterial hypertension. In particular embodiments, a subject may bepre-screened to identify whether they have a mutation that effects BMPR2signaling, or they may be assayed to determine if they have abnormalexpression of Id1.

FK-506 (also known Tacrolimus or Fujimycin) is an immunosuppressive drugthat is mainly used after allogeneic organ transplant to reduce theactivity of the patient's immune system and so lower the risk of organrejection. It is also used for the treatment of severe atopic dermatitis(eczema), severe refractory uveitis after bone marrow transplants, andthe skin condition vitiligo. FK-506 is a 23-membered macrolide lactonediscovered in 1984.

In T-cells, activation of the T-cell receptor normally increasesintracellular calcium, which acts via calmodulin to activatecalcineurin. Calcineurin then dephosphorylates the transcription factorNF-AT (nuclear factor of activated T-cells), which moves to the nucleusof the T-cell and increases the activity of genes coding for IL-2 andrelated cytokines. FK-506 prevents the dephosphorylation of NF-AT.Specifically, FK-506 reduces peptidyl-prolyl isomerase activity bybinding to the immunophilin FKBP12 (FK506 binding protein) creating anew complex. This FKBP12-FK506 complex interacts with and inhibitscalcineurin thus inhibiting both T-lymphocyte signal transduction andIL-2 transcription. In some embodiments, the FK506 is administered at adose and regimen that provides an FK506 serum concentration that is muchlower than the FK506 serum concentration commonly used inimmunosuppressive applications (which is typically 5-15 ng/ml). Forexample, in certain embodiments of the instant method, the FK506 isadministered at a dose and regimen that provides an FK506 serumconcentration of as 0.05 ng/ml to 1 ng/ml, e.g., 0.1 ng/ml to 0.5 ng/ml,0.15 ng/ml to 0.3 ng/ml or e.g. 0.1-0.2 ng/ml. In part because FK-506 ismetabolized by the cytochrome P450 system, the exact dosing may varybetween patients. The FK506 may be administered once a day or more,e.g., twice per day. In immunosuppressive applications, FK506 isnormally given twice daily with the goal to reach FK-506 serum levels of5-15 ng/ml. The treatment is started at 0.5 mg twice daily and thenup-titrated according to the measured FK506 serum level. In some cases adosing of 0.075 mg/kg/day is recommended to reach a serum levels of 5-10ng/ml. In some embodiments of the instant method, the goal is to reach aserum level of about 0.2 ng/ml, which is about 1/20 of theimmunosuppressive serum level. In this case, an initial dose of 0.001mg/kg day to 0.01 mg/kg day (e.g., 0.002 mg kg/day to 0.05 mg/kg/day maybe sufficient, and the does can be up-titrated according to the measuredFK506 serum level. The subject may be any mammal, e.g., a human, rat, ormouse, for example. In particular cases, the FK506 may reach a serumconcentration as low as 0.1-0.2 ng/ml (e.g., 0.10 to 0.12, 0.12 to 0.14,0.14 to 0.16, 0.16 to 0.18 or 0.18 to 0.20, however serum aconcentration in the range of 0.2 to 2 ng/ml, e.g., 0.2, 0.5, 1 and 2ng/ml may be acceptable. In particular cases, the FK506 may reach aserum concentration of <1.0, 1.5-2.5, or 3-5 ng/ml.

The FK506 may be administered alone or in combination with other activecompounds that treat or prevent PAH. The other active compound may beadministered at a different time or at the same time as the FK506 and incertain embodiments the FK506 and the other active compound may bepresent in the same formulation, or as separate formulations in the samekit. Exemplary other active compounds that treat PAH include, e.g.,prostacyclin analogues, endothelin receptor antagonists,phosphodiesterase-5 inhibitors, high-dose calcium channel blockers,anticoagulants, diuretics or antiproliferative agents. In particularcases, the other active compound may be, for example, Isordil(isosorbide dinitrate), Revatio (sildenafil), Tracleer (bosentan),Letairis (ambrisentan), Flolan (epoprostenol), Adcirca (tadalafil),Remodulin (treprostinil) Ventavis (iloprost), Tyvaso (treprostinil),Dilatrate-SR (isosorbide dinitrate), Isordil Titradose (isosorbidedinitrate), IsoDitrate (isosorbide dinitrate) or Isochron (isosorbidedinitrate).

Administration of FK506 to a subject may decrease pulmonary arterialpressure by about at least 1 mm Hg, e.g., at least 2 mm Hg, at least 3mm Hg, at least 4 mm Hg, at least 5 mm Hg or at least 10 mm Hg or more,thereby returning the pulmonary arterial pressure to a level that may beconsidered normal for the subject.

In general terms, the FK506 may be administered to the subject in theinstant method in a similar way to how FK506 is administered inimmunosuppressive applications. For example, the FK506 may be present ina pharmaceutically acceptable excipient, and it may be administeredintravenously. Alternatively, it may be administered orally.

Because the FK506 is being administered at a lower dose, its usual sideeffects may be decreased. Typical side effects include infection,cardiac damage, hypertension, blurred vision, liver and kidney problems(tacrolimus nephrotoxicity), hyperkalemia, hypomagnesemia,hyperglycemia, diabetes mellitus, itching, lung damage (sirolimus alsocauses lung damage), and various neuropsychiatric problems such as lossof appetite, insomnia, Posterior reversible encephalopathy syndrome,confusion, weakness, depression, cramps, neuropathy, seizures, tremors,and catatonia.

Pharmaceutical Compositions

A pharmaceutical composition comprising a subject compound may beadministered to a patient alone, or in combination with othersupplementary active agents. The pharmaceutical compositions may bemanufactured using any of a variety of processes, including, withoutlimitation, conventional mixing, dissolving, granulating, dragee-making,levigating, emulsifying, encapsulating, entrapping, and lyophilizing.The pharmaceutical composition can take any of a variety of formsincluding, without limitation, a sterile solution, suspension, emulsion,lyophilisate, tablet, pill, pellet, capsule, powder, syrup, elixir orany other dosage form suitable for administration.

A subject compound may be administered to the host using any convenientmeans capable of resulting in the desired reduction in disease conditionor symptom. Thus, a subject compound can be incorporated into a varietyof formulations for therapeutic administration. More particularly, asubject compound can be formulated into pharmaceutical compositions bycombination with appropriate pharmaceutically acceptable carriers ordiluents, and may be formulated into preparations in solid, semi-solid,liquid or gaseous forms, such as tablets, capsules, powders, granules,ointments, solutions, suppositories, injections, inhalants and aerosols.

Formulations for pharmaceutical compositions are well known in the art.For example, Remington's Pharmaceutical Sciences, by E. W. Martin, MackPublishing Co., Easton, Pa., 19th Edition, 1995, describes exemplaryformulations (and components thereof) suitable for pharmaceuticaldelivery of disclosed compounds. Pharmaceutical compositions comprisingat least one of the subject compounds can be formulated for use in humanor veterinary medicine. Particular formulations of a disclosedpharmaceutical composition may depend, for example, on the mode ofadministration and/or on the location of the infection to be treated. Insome embodiments, formulations include a pharmaceutically acceptablecarrier in addition to at least one active ingredient, such as a subjectcompound. In other embodiments, other medicinal or pharmaceuticalagents, for example, with similar, related or complementary effects onthe affliction being treated can also be included as active ingredientsin a pharmaceutical composition.

Pharmaceutically acceptable carriers useful for the disclosed methodsand compositions are conventional in the art. The nature of apharmaceutical carrier will depend on the particular mode ofadministration being employed. For example, parenteral formulationsusually comprise injectable fluids that include pharmaceutically andphysiologically acceptable fluids such as water, physiological saline,balanced salt solutions, aqueous dextrose, glycerol or the like as avehicle. For solid compositions (e.g., powder, pill, tablet, or capsuleforms), conventional non-toxic solid carriers can include, for example,pharmaceutical grades of mannitol, lactose, starch, or magnesiumstearate. In addition to biologically neutral carriers, pharmaceuticalcompositions to be administered can optionally contain minor amounts ofnon-toxic auxiliary substances (e.g., excipients), such as wetting oremulsifying agents, preservatives, and pH buffering agents and the like;for example, sodium acetate or sorbitan monolaurate. Other non-limitingexcipients include, nonionic solubilizers, such as cremophor, orproteins, such as human serum albumin or plasma preparations.

Some examples of materials which can serve aspharmaceutically-acceptable carriers include: (1) sugars, such aslactose, glucose and sucrose; (2) starches, such as corn starch andpotato starch; (3) cellulose, and its derivatives, such as sodiumcarboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4)powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients,such as cocoa butter and suppository waxes; (9) oils, such as peanutoil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil andsoybean oil; (10) glycols, such as propylene glycol; (11) polyols, suchas glycerin, sorbitol, mannitol, and polyethylene glycol; (12) esters,such as ethyl oleate and ethyl laurate; (13) agar; (14) bufferingagents, such as magnesium hydroxide and aluminum hydroxide; (15) alginicacid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer'ssolution; (19) ethyl alcohol; (20) pH buffered solutions; (21)polyesters, polycarbonates and/or polyanhydrides; and (22) othernon-toxic compatible substances employed in pharmaceutical formulations.

The disclosed pharmaceutical compositions may be formulated as apharmaceutically acceptable salt of a disclosed compound.Pharmaceutically acceptable salts are non-toxic salts of a free baseform of a compound that possesses the desired pharmacological activityof the free base. These salts may be derived from inorganic or organicacids. Non-limiting examples of suitable inorganic acids arehydrochloric acid, nitric acid, hydrobromic acid, sulfuric acid,hydroiodic acid, and phosphoric acid. Non-limiting examples of suitableorganic acids are acetic acid, propionic acid, glycolic acid, lacticacid, pyruvic acid, malonic acid, succinic acid, malic acid, maleicacid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamicacid, mandelic acid, methanesulfonic acid, ethanesulfonic acid,p-toluenesulfonic acid, methyl sulfonic acid, salicylic acid, formicacid, trichloroacetic acid, trifluoroacetic acid, gluconic acid,asparagic acid, aspartic acid, benzenesulfonic acid, p-toluenesulfonicacid, naphthalenesulfonic acid, and the like. Lists of other suitablepharmaceutically acceptable salts are found in Remington'sPharmaceutical Sciences, 17th Edition, Mack Publishing Company, Easton,Pa., 1985. A pharmaceutically acceptable salt may also serve to adjustthe osmotic pressure of the composition.

A subject compound can be used alone or in combination with appropriateadditives to make tablets, powders, granules or capsules, for example,with conventional additives, such as lactose, mannitol, corn starch orpotato starch; with binders, such as crystalline cellulose, cellulosederivatives, acacia, corn starch or gelatins; with disintegrators, suchas corn starch, potato starch or sodium carboxymethylcellulose; withlubricants, such as talc or magnesium stearate; and if desired, withdiluents, buffering agents, moistening agents, preservatives andflavoring agents. Such preparations can be used for oral administration.

A subject compound can be formulated into preparations for injection bydissolving, suspending or emulsifying them in an aqueous or nonaqueoussolvent, such as vegetable or other similar oils, synthetic aliphaticacid glycerides, esters of higher aliphatic acids or propylene glycol;and if desired, with conventional additives such as solubilizers,isotonic agents, suspending agents, emulsifying agents, stabilizers andpreservatives. The preparation may also be emulsified or the activeingredient encapsulated in liposome vehicles. Formulations suitable forinjection can be administered by an intravitreal, intraocular,intramuscular, subcutaneous, sublingual, or other route ofadministration, e.g., injection into the gum tissue or other oraltissue. Such formulations are also suitable for topical administration.

In some embodiments, a subject compound can be delivered by a continuousdelivery system. The term “continuous delivery system” is usedinterchangeably herein with “controlled delivery system” and encompassescontinuous (e.g., controlled) delivery devices (e.g., pumps) incombination with catheters, injection devices, and the like, a widevariety of which are known in the art.

A subject compound can be utilized in aerosol formulation to beadministered via inhalation. A subject compound can be formulated intopressurized acceptable propellants such as dichlorodifluoromethane,propane, nitrogen and the like.

Furthermore, a subject compound can be made into suppositories by mixingwith a variety of bases such as emulsifying bases or water-solublebases. A subject compound can be administered rectally via asuppository. The suppository can include vehicles such as cocoa butter,carbowaxes and polyethylene glycols, which melt at body temperature, yetare solidified at room temperature.

The term “unit dosage form,” as used herein, refers to physicallydiscrete units suitable as unitary dosages for human and animalsubjects, each unit containing a predetermined quantity of a subjectcompound calculated in an amount sufficient to produce the desiredeffect in association with a pharmaceutically acceptable diluent,carrier or vehicle. The specifications for a subject compound depend onthe particular compound employed and the effect to be achieved, and thepharmacodynamics associated with each compound in the host.

The dosage form of a disclosed pharmaceutical composition will bedetermined by the mode of administration chosen. For example, inaddition to injectable fluids, topical or oral dosage forms may beemployed. Topical preparations may include eye drops, ointments, spraysand the like. Oral formulations may be liquid (e.g., syrups, solutionsor suspensions), or solid (e.g., powders, pills, tablets, or capsules).Methods of preparing such dosage forms are known, or will be apparent,to those skilled in the art.

Certain embodiments of the pharmaceutical compositions comprising asubject compound may be formulated in unit dosage form suitable forindividual administration of precise dosages. The amount of activeingredient administered will depend on the subject being treated, theseverity of the affliction, and the manner of administration, and isknown to those skilled in the art. Within these bounds, the formulationto be administered will contain a quantity of the extracts or compoundsdisclosed herein in an amount effective to achieve the desired effect inthe subject being treated.

Each therapeutic compound can independently be in any dosage form, suchas those described herein, and can also be administered in various ways,as described herein. For example, the compounds may be formulatedtogether, in a single dosage unit (that is, combined together in oneform such as capsule, tablet, powder, or liquid, etc.) as a combinationproduct. Alternatively, when not formulated together in a single dosageunit, an individual subject compound may be administered at the sametime as another therapeutic compound or sequentially, in any orderthereof.

Methods of Administration

The route of administration may be selected according to a variety offactors including, but not necessarily limited to, the condition to betreated, the formulation and/or device used, the patient to be treated,and the like. Routes of administration useful in the disclosed methodsinclude but are not limited to oral and parenteral routes, such asintravenous (iv), intraperitoneal (ip), rectal, topical, ophthalmic,nasal, and transdermal. Formulations for these dosage forms aredescribed herein.

An effective amount of a subject compound will depend, at least, on theparticular method of use, the subject being treated, the severity of theaffliction, and the manner of administration of the therapeuticcomposition. A “therapeutically effective amount” of a composition is aquantity of a specified compound sufficient to achieve a desired effectin a subject (host) being treated.

Therapeutically effective doses (or growth inhibitory amounts) of asubject compound or pharmaceutical composition can be determined by oneof skill in the art, with a goal of achieving local (e.g., tissue)concentrations that are at least as high as the IC50 of an applicablecompound disclosed herein.

The specific dose level and frequency of dosage for any particularsubject may be varied and will depend upon a variety of factors,including the activity of the subject compound, the metabolic stabilityand length of action of that compound, the age, body weight, generalhealth, sex and diet of the subject, mode and time of administration,rate of excretion, drug combination, and severity of the condition ofthe host undergoing therapy.

In order to further illustrate the present invention, the followingspecific examples are given with the understanding that they are beingoffered to illustrate the present invention and should not be construedin any way as limiting its scope.

EXAMPLE I Mouse Study Background

A loss of function mutation in bone morphogenetic protein (BMP) receptorII (BMPRII) is present in >80% of familial and ˜20% of sporadicidiopathic (I) PAH (Machado et al. Hum Mutat 2006, 27:121-32). Evenpatients with IPAH without a BMPRII mutation or with other causes of PAHhave reduced expression of BMPRII, reinforcing the importance of BMPRIIsignaling in the pathogenesis of PAH (Humbert M et al. Eur Respir J2002, 20:518-23). Furthermore BMPR2 receptor gene therapy attenuatesexperimental hypoxic pulmonary hypertension in rats (Reynolds et al. AmJ Physiol Lung Cell Mol Physiol 2007). Therefore increasing BMPRIIsignaling in patients with pulmonary arterial hypertension might preventor reverse disease.

Methods

3600 FDA approved drugs and bioactive compounds were screened for theirability to activate BMP signaling, using a C2C12 mouse myoblast cellline stably transfected with a reporter plasmid expressing a BMPresponse element (BRE) from the Id1 promoter fused to theluciferase-gene (BRE-luc). Whether the best qHTS-BMPRII activator caninduce Smad phosphorylation (phospho), Id1 expression and promote PAECsurvival and tube formation was determined using BMP4 as a positivecontrol. Whether the qHTS-BMPRII-activator would prevent PAH in micewith a conditional deletion in BMPRII in ECs (BMPRII-SCL-CreERT) thatdevelop exaggerated PAH after 3 weeks of hypoxia (10% O₂) wasdetermined. In order to assess whether the BMPRII activator could alsoreverse PAH, we used 2 models of severe experimental PH in rats: 1.Monocrotaline induced pulmonary hypertension with development of severemedial hypertrophy of the pulmonary arteries 3 weeks after injection. 2.SUGEN (VEGF-Receptor blocker) and 3-week chronic hypoxia inducedpulmonary hypertension with development of neointima formation inpulmonary arteries 8 weeks after initiation of the stimulus. Both groupswere treated with FK-506 for 3 weeks via sc osmotic pump (0.05 mg/kg/d)after PAH and remodeling of the pulmonary arteries was established. Theserum level of FK-506 in mice and rats was aimed to be 0.2 ng/ml.

Results

FK-506, an agent that can induce BMPRIA phosphorylation, was the mainactivator of Id1 expression. FK-506, at a dose of 15 ng/ml, thetherapeutic serum level used to induce immunosuppression, and at a muchlower dose of 0.2 ng/ml increased Id1 protein expression 1 h followingstimulation, in a manner comparable to BMP4 (10 ng/ml) (n=3, p<0.06).This was preceded by phospho-Smad 1/5/8 at 15 min, similar to BMP4 (n=3,p<0.001). FK-506 induced p-Smad 1/5/8 and Id1 expression in PAECsharvested from six different IPAH patients at the time of transplant,including 3/6 patients that did not respond to BMP4. Both BMP4 andFK-506 improved survival of PAECs (n=5, p<0.001) and induced tubeformation in an angiogenesis assay (n=3, p<0.01). A 3-week preventivetreatment with FK-506 (0.05 mg/kg/d) (serum levels 0.2 ng/ml) in micewith a conditional deletion in BMPRII in ECs exposed to 3 weeks ofhypoxia prevented the development of PAH and right ventricularhypertrophy (RVH); RV systolic pressure: 32±0.9 vs 21±2.3 mmHg, p<0.001;RVH: 36.2±2.5 vs 26.9±4.5, p<0.01, both n=5. To test whether FK-506could also reverse PAH, we induced PAH in rats with monocrotaline (60 mgonce s.c) and began treatment with FK-506 3 weeks after injection, atime when PAH was established (RVSP 50.8±2.7 mmHg, n=7). The survivalafter a 3-week treatment with FK-506 did not differ in the FK-506 (57%)compared to the vehicle group (66%), yet of those that that survived thePAH was significantly reduced after treatment with FK-506 compared tovehicle treated animals (RVSP 39.5 ±4.7 vs 68.6 ±4.2 mmHg, n=14).

It was determined that the combined stimulus of SUGEN (20 mg/kg s.c) and3-weeks of chronic hypoxia induced PAH in rats when rats were returnedto RA and left for another 5 weeks (RVSP 55.1±10.7 vs control 25.1±0.5mmHg, RVH 0.24±0.005 vs 0.44±0.07, n=4, p<0.05) but that a 3-week sctreatment of FK-506 at the time of established PAH could preventprogression and induce regression of PAH in FK-506 treated vs vehicletreated animals (RVSP 66.5±4.1 mmHg vs 39.5±0.6 mmHg, RVH 0.49±0.07 vs0.34±0.02, n=4, p<0.05). Neointima formation in small pulmonary arteries(alveolar wall and alveolar duct vessels) per total vessel numberdecreased from 61.2±6.1% to 16.2±5.8% (n=4, p<0.01). At the low dose ofFK-506 of 0.2 ng/ml no effect on total or differential WBC count wasobserved, nor was an immunosuppressive effect of decreased nuclearNFATc2 measured.

Conclusion

FK-506 (Tacrolimus) was identified in a quantitative high throughputscreen (qHTS) of FDA approved drugs and bioactive compounds as a drugthat activates BMPRII signaling, restores normal function of pulmonaryartery endothelial cells (PAECs), prevents and reverses experimental PAHin mice and rats.

EXAMPLE 2 Clinical Study

Patients are invited to participate in this study because they havepulmonary hypertension (PH) and are currently treated with one ormultiple drugs for PH such as PDE-5 inhibitors (sildenafil, tadalafil),prostacyclins (Flolan, Remodulin, Iloprost) and/or the endothelinantagonist Ambrisentan. While all these drugs are effective asvasodilators, new medications are sought that could reverse thepathological remodeling of the pulmonary arteries. Whether subjects havea familial form of pulmonary hypertension or not, it is known that acertain pathway (BMPR2) is impaired in PH. Studies have shown that theimmunosuppressive drug FK-506 (Tacrolimus) activates the BMPR2 pathwayand prevents and reverses pulmonary hypertension in experimentalpulmonary hypertension.

This study is open to male or female subjects, 18-70 years of age, withPH. If a patient agrees to participate in this study, the patient willbe one of 40 subjects participating in the study.

If a patient agrees to participate and the patent qualifies, the patientwill be allocated to the study drug through a process calledrandomization. Randomization means that the study drug that the patientwill receive is selected by chance (like the flip of a coin). The studydrug options for this study are placebo, and 3 different doses of FK-506(blood level<1.0, 1.5-2.5, and 3-5 ng/ml; as a reference: theimmunosuppressive dose is 5-15 ng/ml). The study drug will be added tothe patient's baseline PH therapy. The randomization for this study is1:3 which means patients have a chance of 75% of receiving treatmentwith FK-506.

The purpose of this study is to confirm that adding FK-506 to a PHtreatment at a dose below the normal dose that is used forimmunosuppression is safe and whether it will improve pulmonaryhypertension. Heart function will be assessed by echo, 6-min walk andthe biomarker NT-proBNP.

Study Medication FK-506 (Tacrolimus) is an FDA approvedimmunosuppressive drug used in organ transplantation as well as inautoimmune diseases. As the metabolism of FK-506 differs in patientsquite widely, therapy is directed by measuring drug levels in wholeblood. The blood will be drawn shipped to a testing lab to measureFK-506 levels. The goal immunosuppressive doses are 5-15 ng/ml. In thisstudy we aim for much lower doses (see above). Patients will receive thestudy drug for the duration of study. The drug will be delivered in aprepared bottle, which allows monitoring of drug intake. This device iscalled a Medication Event Monitoring System (MEMS) and for it to monitordrug intake properly. Patients should always take out one tablet at atime from the bottle.

Participation in the study lasts for approximately 16 weeks. During thistime, patients will be required to visit the clinic approximately 4-5times.

Study Procedures

If a patient agrees to take part in this study, they will first signthis consent form. After the patients have signed, dated and received acopy of this consent form, they will have the study screening visit toensure the patient is eligible to take part in this study. Previous testresults (echocardiogram, physical examination, pulmonary function tests,Right Heart Catheterization (RHC) may also be used to determine patienteligibility.

1-8. (canceled)
 9. A method of treating pulmonary arterial hypertensionin a patient in need thereof, the method comprising: administering FK506to the patient at a daily dosage of 0.002 mg/kg of the patient's weightto 0.05 mg/kg of the patient's weight.
 10. The method of claim 9,wherein the dosage provides an FK506 blood concentration of 0.05 ng/mLto 1 ng/mL.
 11. The method of claim 9, wherein the dosage provides anFK506 blood concentration of 0.1 ng/mL to 0.2 ng/mL.
 12. The method ofclaim 9, wherein the dosage provides an FK506 blood concentration of 0.2ng/mL to 2 ng/mL.
 13. The method of claim 9, wherein the dosage providesan FK506 blood concentration of 1.5 ng/mL to 2.5 ng/mL.
 14. The methodof claim 9, wherein the dosage provides an FK506 blood concentration of3 ng/mL to 5 ng/mL.
 15. The method of claim 9, wherein the dosageprovides an FK506 blood concentration of FK506 of below 1 ng/mL.
 16. Themethod of claim 9, further comprising administering the FK506 incombination with a therapeutically effective amount of at least oneadditional active compound.
 17. The method of claim 16 where the atleast one additional active compound is one or more selected from thefollowing list: sildenafil, bosentan, ambrisentan, epoprostenol,tadalafil, treprostinil iloprost, treprostinil, isosorbide dinitrate, orisosorbide dinitrate.
 18. The method of claim 16 where the at least oneadditional active compound is one or more selected from the followinglist: prostacyclin analogues, endothelin receptor antagonists,phosphodiesterase-5 inhibitors, high-dose calcium channel blockers,antigoagulants, diuretics and antiproliferative agents.
 19. A method oftreating pulmonary arterial hypertension in a patient in need thereof,the method comprising: administering FK506 to the patient at a dailydosage of 0.001 mg/kg of the patient's weight to 0.01 mg/kg of thepatient's weight.
 20. The method of claim 19, wherein the dosageprovides an FK506 blood concentration of 0.05 ng/mL to 1 ng/mL.
 21. Themethod of claim 19, wherein the dosage provides an FK506 bloodconcentration of FK506 of below 1 ng/mL.